阅读说明：本技术 一种可用于预防、治疗非酒精性脂肪肝的产品 (A product for preventing and treating non-alcoholic fatty liver disease ) 是由 王刚 赵文宇 顾震南 翟齐啸 陆文伟 赵建新 张灏 陈卫 于 2020-07-29 设计创作，主要内容包括：本发明公开了一种可用于预防、治疗非酒精性脂肪肝的产品,属于生物医药技术领域。本发明制备的肉豆蔻提取物能够抑制肝脏细胞LO2脂肪酸的合成、抑制肝脏细胞LO2脂肪酸合成酶基因FASN,固醇调节元件结合蛋白基因SREBP-1c的表达,改善肝功能,调节脂代谢,改善肝脏炎症反应,增强小鼠免疫功能,调节肝脏脂质代谢,降低肝脏脂质堆积,有助于预防和/或非酒精性脂肪肝。(The invention discloses a product for preventing and treating non-alcoholic fatty liver disease, and belongs to the technical field of biological medicine. The myristica fragrans extract prepared by the method can inhibit synthesis of fatty acid LO2 of liver cells, inhibit expression of fatty acid synthase gene FASN of LO2 of liver cells and sterol regulatory element binding protein gene SREBP-1c, improve liver functions, regulate lipid metabolism, improve liver inflammatory response, enhance immune functions of mice, regulate liver lipid metabolism, reduce liver lipid accumulation and help prevent and/or treat nonalcoholic fatty liver.)

1. The application of the myristica fragrans extract in preparing products for preventing and/or treating non-alcoholic fatty liver diseases is characterized in that the myristica fragrans extract is prepared by the following steps:

(1) pulverizing the roasted semen Myristicae, grinding, and sieving;

(2) leaching the sieved nutmeg powder in the step (1) for at least 10 hours by using an ethanol solution, and repeating for 2-3 times;

(3) mixing the extracts, vacuum filtering, and concentrating to obtain semen Myristicae extract.

2. The use according to claim 1, wherein the myristica fragrans extract is dissolved in a solvent.

3. Use according to claim 1 or 2, wherein the product comprises but is not limited to a food, a pharmaceutical or a nutraceutical product.

4. Use according to any one of claims 1 to 3, wherein the prevention and/or treatment of non-alcoholic fats is at least one of (a) to (f):

(a) reducing inflammatory response caused by free fatty acids;

(b) inhibiting hepatocyte lipid synthesis and macrophage inflammatory response;

(c) reducing blood fat;

(d) reducing blood glucose and improving glucose tolerance;

(e) reducing liver lipid synthesis;

(f) regulating liver lipid metabolism and/or reducing liver lipid accumulation;

(g) improving liver function;

(h) inhibit the proliferation of liver cancer cell and reduce the survival rate of cancer cell.

5. A pharmaceutical composition comprising an extract of Myristica fragrans Houtt; the nutmeg extract is prepared by the following steps:

(1) pulverizing the roasted semen Myristicae, grinding, and sieving;

(2) leaching the sieved nutmeg powder in the step (1) for at least 10 hours by using an ethanol solution, and repeating for 2-3 times;

(3) mixing the extracts obtained in step (2), vacuum filtering, and concentrating to obtain semen Myristicae extract.

6. The pharmaceutical composition of claim 5, wherein the myristyl alcohol extract is present in an amount of no less than 200 mg/kg.

7. The pharmaceutical composition of claim 5 or 6, further comprising a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the carrier is one or more of a pharmaceutically acceptable filler, wetting agent, disintegrant, binder, or lubricant.

9. The pharmaceutical composition of claim 8, wherein the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

10. Use of Myristica fragrans Houtt extract in the preparation of non-medical health products.

Technical Field

The invention relates to a product for preventing and treating non-alcoholic fatty liver, belonging to the technical field of biological medicine.

Background

Non-alcoholic fatty liver disease is a disease characterized by excessive accumulation of fat in hepatocytes. It is usually associated with severe obesity and chronic low grade inflammation. The clinicopathological syndrome of nonalcoholic fatty liver disease is characterized by diffuse hepatocytes with hepatocellular globulocystic, intrahepatic inflammation and progressive fibrosis. According to the pathological changes of the non-alcoholic fatty liver, the liver can be divided into simple steatosis, non-alcoholic steatohepatitis and fatty liver cirrhosis, and fibrosis even develops into liver cancer. Persistent liver fatty infiltration and inflammatory responses can increase the incidence of cirrhosis and liver cancer. In addition, non-alcoholic fatty liver promotes the development of dyslipidemia and significantly increases the incidence of cardiovascular disease. The mechanism by which the non-alcoholic fatty liver process occurs is not clear, although it is generally believed that it consists of at least two components, the "secondary strike" theory. Liver fat accumulation caused by disorders of fat metabolism is a common mechanism among the various causes causing liver steatosis, followed by immune cell activation and inflammatory cytokine production.

Studies have shown that obese patients with non-alcoholic fatty liver have decreased lipid metabolism but increased fatty acid synthesis. The fatty acid synthase gene (FASN) and the upstream sterol regulatory element binding protease-1 c (SREBP-1c) play an important role in fatty acid metabolism, activation of upstream phosphoadenosine protease (AMPK) accelerates energy metabolism, and inhibition of FASN leads to a decrease in the fatty acid synthesis pathway. Therefore, the method has become a powerful target for intervening the non-alcoholic fatty liver caused by excessive lipid accumulation. Sustained excess of free fatty acids is involved in the pathogenesis of steatosis and leads to non-alcoholic fatty liver-related metabolic complications, such as inflammatory reactions. Prolonged exposure of the liver to excess free fatty acids can lead to accumulation of intracellular triglycerides and impair insulin function, further affecting blood glucose levels. Excessive fatty acids promote TG secretion in the form of VLDL and increase hepatic neolipogenesis, leading to hepatotoxicity and inducing the liver to continuously produce inflammatory factors, affecting liver function. By inhibiting fatty acid synthesis, the proliferation and growth of liver cancer cells can be inhibited, and the survival rate is reduced. Therefore, the lipid metabolism is regulated to become a powerful means for losing weight and lowering lipid, inhibiting inflammatory reaction, recovering liver function and improving non-alcoholic fatty liver.

At present, non-alcoholic fatty liver disease can be prevented to some extent by limiting dietary energy intake (e.g. low sugar, low fat, etc.) or by exercising in moderate amounts, but the extent of intervention is limited. With the acceleration of modern life rhythm and the increase of social working pressure, irregular diet and sleep increase the incidence of the non-alcoholic fatty liver disease. However, the drug therapy is accompanied by a series of side effects, and the drug therapy causes a certain degree of damage to the body. Therefore, there is a need to develop a product for preventing and treating non-alcoholic fatty liver disease to solve the problem.

Disclosure of Invention

The invention aims to solve the technical problems of providing a novel health care food which can reduce weight, blood fat and liver lipid accumulation; can also regulate inflammatory reaction and reduce the generation of complications; and has little toxic and side effects, and can be used for preventing/treating non-alcoholic fatty liver.

The invention provides application of a myristica fragrans extract in preparation of a product for preventing/treating non-alcoholic fatty liver disease.

In one embodiment, the myristica fragrans extract is prepared by the following steps:

(1) pulverizing the roasted semen Myristicae, grinding, and sieving;

(2) leaching the sieved nutmeg powder in the step (1) for at least 10 hours by using an ethanol solution, and repeating for 2-3 times;

(3) mixing the extracts, vacuum filtering, and concentrating to obtain semen Myristicae extract.

In one embodiment, the myristica fragrans extract is dissolved in a solvent.

In one embodiment, the solvent includes, but is not limited to, dimethyl sulfoxide (DMSO) or sodium carboxymethyl cellulose (CMC) with a mass fraction of 0.5%.

In one embodiment, the myristica fragrans extract is prepared as follows:

(1) pulverizing the roasted nutmeg, grinding with a mechanical grinder, and sieving with a 200-mesh sieve;

(2) leaching the sieved nutmeg powder in the step (1) with 75% ethanol at 37 ℃ for 12h, filtering and recovering dregs, and repeating the leaching step for 3 times;

(3) mixing the 3 times of extracts, and vacuum filtering;

(4) concentrating the filtered liquid in a rotary evaporator, evaporating to dryness, and dissolving in a solvent; the solvent includes but is not limited to dimethyl sulfoxide (DMSO) or sodium carboxymethyl cellulose (CMC) with a mass fraction of 0.5%.

In one embodiment, the prevention/treatment of non-alcoholic fatty dryness is embodied in at least one of (a) to (f):

(a) reducing inflammatory response caused by free fatty acids;

(b) inhibiting hepatocyte lipid synthesis and macrophage inflammatory response;

(c) reducing blood fat;

(d) reducing blood glucose and improving glucose tolerance;

(e) reducing liver lipid synthesis;

(f) regulating liver lipid metabolism and/or reducing liver lipid accumulation;

(g) improving liver function;

(h) inhibit the proliferation of liver cancer cell and reduce the survival rate of cancer cell.

In one embodiment, the product is a functional food.

The second object of the present invention is to provide a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, which comprises the aforementioned myristica fragrans extract.

In one embodiment, the myristica fragrans extract is dissolved in an ethanol solution, dimethyl sulfoxide (DMSO) or a 0.5% by weight solution of sodium carboxymethylcellulose (CMC).

In one embodiment, the myristica fragrans extract is present in the pharmaceutical composition in an amount of not less than 200 mg/kg.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In one embodiment, the carrier is one or more of a pharmaceutically acceptable filler, wetting agent, disintegrant, binder, or lubricant.

In one embodiment, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

The invention also claims the use of the nutmeg extract in the preparation of a health product for non-medical purposes.

Has the advantages that:

(1) the invention takes nutmeg as RAW material to prepare nutmeg extract which can inhibit the synthesis of fatty acid of liver cells LO2, reduce inflammatory reaction of mouse macrophage RAW264.7 caused by free fatty acid, and inhibit the expression of mouse liver cells LO2 fatty acid synthetase gene FASN and sterol regulatory element binding protein gene SREBP-1 c. Compared with a control group, the expression quantity of the FASN gene and the SREBP-1c gene is obviously reduced, so that the product has better inhibition effect on in-vitro mouse hepatocyte lipid synthesis and macrophage inflammatory reaction.

(2) Can effectively inhibit the in vitro proliferation of the mouse liver cancer cell Hepa1-6, and is obviously superior to the reported inhibition level.

(3) The myristica fragrans extract disclosed by the invention can be used for reducing the weight of a mouse, reducing blood fat, obviously reducing liver lipid, improving the blood sugar level and the liver function and has a strong effect of preventing and/or treating nonalcoholic fatty liver.

(4) After the mouse is subjected to intragastric administration, the content of inflammatory factors IL-6 and TNF alpha in the liver of the mouse after the myristica fragrans extract prepared by the method is obviously reduced at 16 weeks of a non-alcoholic fatty liver disease model. Therefore, the myristica fragrans extract can improve the liver inflammatory reaction and enhance the immune function of mice.

(5) Protein expression levels of FASN and SREBP-1c in the mouse liver after the maceration of the myristica fragrans extract prepared by the method are obviously reduced compared with a model control group mouse, and the expression levels of pAMPK-alpha/AMPK-alpha are obviously increased compared with the control group mouse.

Drawings

Figure 1 effect of different myristol extract concentrations on LO2 hepatocyte survival.

FIG. 2 the effect of different concentrations of myristyl alcohol extract on the FASN and SREBP-1c genes of LO2 hepatocytes.

FIG. 3 shows the effect of different concentrations of myristyl alcohol extract on the expression level of FASN gene in LO2 hepatocytes.

FIG. 4 shows the effect of different concentrations of myristyl alcohol extract on the expression level of SREBP-1c gene of LO2 hepatocytes.

FIG. 5 shows the effect of myristyl alcohol extract on the expression level of TNF alpha gene, a mouse macrophage inflammatory factor.

FIG. 6 shows the effect of myristyl alcohol extract on the expression level of mouse macrophage inflammatory factor IL-6 gene.

FIG. 7 effect of myristyl alcohol extract on food intake of non-alcoholic fatty liver disease model mice.

FIG. 8 the effect of myristyl alcohol extract on body weight of non-alcoholic fatty liver disease model mice.

FIG. 9 the effect of myristyl alcohol extract on the amount of weight change in non-alcoholic fatty liver disease model mice.

FIG. 10 effect of myristyl alcohol extract on blood glucose levels in non-alcoholic fatty liver disease model mice.

FIG. 11 Effect of myristyl alcohol extract on oral glucose tolerance curves in non-alcoholic fatty liver disease model mice.

Figure 12 effect of myristyl alcohol extract on area under oral glucose tolerance curve of non-alcoholic fatty liver disease model mice.

FIG. 13 effect of myristyl alcohol extract on liver lipid accumulation in non-alcoholic fatty liver model mice.

FIG. 14 Effect of myristyl alcohol extract on serum low density lipoprotein levels in non-alcoholic fatty liver model mice.

FIG. 15 effect of myristyl alcohol extract on serum high density lipoprotein levels in non-alcoholic fatty liver disease model mice.

FIG. 16 Effect of myristyl alcohol extract on serum cholesterol levels in non-alcoholic fatty liver model mice.

FIG. 17 Effect of myristyl alcohol extract on serum alanine aminotransferase levels in non-alcoholic fatty liver model mice.

FIG. 18 effect of myristyl alcohol extract on serum alkaline phosphatase levels in non-alcoholic fatty liver model mice.

Figure 19 effect of myristyl alcohol extract on the level of inflammatory factor TNF α in the liver of non-alcoholic fatty liver model mice.

FIG. 20 Effect of myristyl alcohol extract on the level of inflammatory factor IL-6 in the liver of non-alcoholic fatty liver model mice.

FIG. 21 shows the effect of myristyl alcohol extract on the relative expression level of FASN gene in liver of non-alcoholic fatty liver disease model mouse.

FIG. 22 shows the effect of myristyl alcohol extract on the relative expression level of SREBP-1c gene in liver of non-alcoholic fatty liver disease model mouse.

FIG. 23 shows the effect of myristyl alcohol extract on the expression level of FASN, SREBP-1c and β -actin in liver of non-alcoholic fatty liver disease model mouse.

FIG. 24 Effect of myristyl alcohol extracts on pAMPK- α/AMPK- α in the liver of non-alcoholic fatty liver model mice.

(P < 0.05; P < 0.01; significance is represented by abc: no significant difference between identical letters, significant difference between different letters.)

Detailed Description

Nutmeg referred to in the following examples was purchased from Tongrentang, Beijing;